Polymorphic forms of (S).-tetrahydrofuran-3-yl-3-(3-(3-methoxy-4-(oxazol-5-yl) phenyl) ureido) benzylcarbamate

ABSTRACT

The present invention relates to polymorphic forms of (S)-tetrahydrofuran-3-yl 3-(3-(3-methoxy-4-(oxazol-5-yl)phenyl)ureido)benzylcarbamate (Compound 1): 
                         
and pharmaceutical compositions thereof. The present invention also relates to processes to prepare compound 1 and pharmaceutical compositions thereof. Compound 1 is an IMPDH inhibitor useful in treating IMPDH-mediated diseases such as immune system related diseases, viral diseases, and cancers.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119 of U.SProvisional application Ser. No. 60/679,021 filed May 9, 2005, theentire contents of which is incorporated herein by reference.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to polymorphic forms of(S)-tetrahydrofuran-3-yl3-(3-(3-methoxy-4-(oxazol-5-yl)phenyl)ureido)benzylcarbamate, processestherein, pharmaceutical compositions thereof, and methods therewith.

BACKGROUND OF THE INVENTION

The present invention relates to polymorphic forms of(S)-tetrahydrofuran-3-yl3-(3-(3-methoxy-4-(oxazol-5-yl)phenyl)ureido)benzylcarbamate having thestructure below (hereinafter “Compound 1”):

The present invention also relates to processes to prepare polymorphicforms of Compound 1.

Compound 1 is a potent IMPDH inhibitor useful in treating IMPDH-mediateddiseases. Compound 1, compositions thereof, and methods therewith aredisclosed in U.S. Pat. Nos. 5,807,876; 6,054,472; 6,344,465; and6,541,496 (hereinafter “the Compound 1 patents”), the entire disclosuresof which are incorporated herein by reference.

SUMMARY OF THE INVENTION

The present invention provides five polymorphic forms of Compound 1,namely, Form A1, Form B2, Form C3, Form D4, and Form E5. The presentinvention also relates to processes for making these polymorphic forms.The invention also relates to the use of these polymorphic forms intherapeutic methods and in the preparation of pharmaceuticalcompositions comprising such polymorphic forms. The present inventionalso relates to an amorphous form of Compound 1, and processes forproducing such an amorphous form.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is an x-ray powder diffraction pattern (XRPD) of Form A1 of(S)-tetrahydrofuran-3-yl3-(3-(3-methoxy-4-(oxazol-5-yl)phenyl)ureido)benzylcarbamate.

FIG. 2 is an XRPD of Form B2 of (S)-tetrahydrofuran-3-yl3-(3-(3-methoxy-4-(oxazol-5-yl)phenyl)ureido)benzylcarbamate.

FIG. 3 is an XRPD of Form C3 of (S)-tetrahydrofuran-3-yl3-(3-(3-methoxy-4-(oxazol-5-yl)phenyl)ureido)benzylcarbamate.

FIG. 4 is an XRPD of Form D4 of (S)-tetrahydrofuran-3-yl3-(3-(3-methoxy-4-(oxazol-5-yl)phenyl)ureido)benzylcarbamate, HCl salt.

FIG. 5 is an XRPD of Form E5 of (S)-tetrahydrofuran-3-yl3-(3-(3-methoxy-4-(oxazol-5-yl)phenyl)ureido)benzylcarbamate, HCl salt.

FIG. 6 is a combination Differential Scanning Calorimetry (DSC)thermogram and thermogravimetric analysis (TGA) of Form D4 of(S)-tetrahydrofuran-3-yl3-(3-(3-methoxy-4-(oxazol-5-yl)phenyl)ureido)benzylcarbamate, HCl salt.

FIG. 7 is a DSC thermogram of Form E5 of (S)-tetrahydrofuran-3-yl3-(3-(3-methoxy-4-(oxazol-5-yl)phenyl)ureido)benzylcarbamate, HCl salt.

FIG. 8 is a DSC thermogram of Form A1 of (S)-tetrahydrofuran-3-yl3-(3-(3-methoxy-4-(oxazol-5-yl)phenyl)ureido)benzylcarbamate.

FIG. 9 is a DSC thermogram of Form B2 of (S)-tetrahydrofuran-3-yl3-(3-(3-methoxy-4-(oxazol-5-yl)phenyl)ureido)benzylcarbamate.

FIG. 10 is a DSC thermogram of Form C3 of (S)-tetrahydrofuran-3-yl3-(3-(3-methoxy-4-(oxazol-5-yl)phenyl)ureido)benzylcarbamate.

FIG. 11 is an XRPD of amorphous (S)-tetrahydrofuran-3-yl3-(3-(3-methoxy-4-(oxazol-5-yl)phenyl)ureido)benzylcarbamate.

FIG. 12 is a DSC thermogram of amorphous (S)-tetrahydrofuran-3-yl3-(3-(3-methoxy-4-(oxazol-5-yl)phenyl)ureido)benzylcarbamate.

DETAILED DESCRIPTION OF THE INVENTION

(S)-tetrahydrofuran-3-yl3-(3-(3-methoxy-4-(oxazol-5-yl)phenyl)ureido)benzylcarbamate(Compound 1) is a potent IMPDH inhibitor useful in treatingIMPDH-mediated diseases. Three polymorphic forms of the free base ofCompound 1 (Form A1, Form B2, and Form C3) have been identified. Twopolymorphic forms of an HCl salt of Compound 1 (Form D4 and Form E5)have also been identified.

The polymorphs of the present invention may occur as racemates, racemicmixtures, and diastereomeric mixtures with all possible isomers andmixtures thereof being included in the present invention.

According to one embodiment, the present invention provides polymorphicForm A1, Form B2, Form C3, Form D4, and Form E5 of Compound 1 with the(S) stereochemistry as indicated in the structure of Compound 1 herein.

According to one embodiment, the present invention provides apolymorphic Form A1 of (S)-tetrahydrofuran-3-yl3-(3-(3-methoxy-4-(oxazol-5-yl)phenyl)ureido)benzylcarbamate.

According to another embodiment, the present invention provides asubstantially pure polymorphic Form A1 of (S)-tetrahydrofuran-3-yl3-(3-(3-methoxy-4-(oxazol-5-yl)phenyl)ureido)benzylcarbamate, whereinsaid polymorph comprises less than about 5% by weight of amorphous form.

According to another embodiment, the present invention provides apolymorphic Form A1 of Compound 1 wherein the polymorph has a peakposition at about 21.8 degrees 2-theta in an x-ray powder diffractionpattern obtained using Cu K alpha radiation.

According to another embodiment, the present invention provides apolymorphic Form A1 of Compound 1 wherein the polymorph has at least oneadditional peak position at about 11.8, 16.0, 18.5, 20.1, or 23.6degrees 2-theta in an x-ray powder diffraction pattern obtained using CuK alpha radiation.

According to another embodiment, the present invention provides apolymorphic Form A1 of Compound 1 wherein the polymorph exhibits amelting/decomposition endothermic event at about 215° C. as measured bya Differential Scanning Calorimeter (DSC). Depending on the rate ofheating or the scan rate at which the DSC analysis is conducted, thecalibration standard used, the relative humidity and upon the chemicalpurity, the endotherms of the respective Forms A1, B2, C3, D4, E5, andamorphous form may vary by about 0.01 to 100° C. above or below theendotherms depicted in the Figures. For any given sample, the observedendotherm may also differ from instrument to instrument; however, itwill generally be within the ranges defined herein provided theinstruments are calibrated similarly.

According to another embodiment, Form A1 is an anhydrous crystallineform with a melting/decomposition temperature of about 215° C. The DSCshows a single endotherm at about 215° C. which corresponds to theon-set of significant weight loss in the thermogravimetric analysis(TGA). Up to this temperature about a 0.4% weight loss was observed.Form A1 is non-hygroscopic with a water uptake of less than 0.5% at 90%relative humidity (RH) at 25° C. Microscopic analysis of Form A1 showedit to contain particles of size 30-75 μm and showed bi-refringence.

According to another embodiment, the present invention provides aprocess for preparing Form A1, as exemplified herein below.

According to another embodiment, the present invention provides aprocess for preparing Form A1 by heating amorphous Compound 1 above itsmelting point to form a molten mass and then cooling said mass whereuponsaid mass recrystallizes to Form A1 between about 120° C. to about 160°C.

According to another embodiment, the present invention provides a methodof converting polymorph Form B2 or Form C3 to Form A1 by exposing neatForm B2 or Form C3 to a suitable elevated temperature for a suitableperiod of time and then cooling to provide Form A1.

In another embodiment, increasing the temperature accelerates thetransformation rate of Form B2 or Form C3 to Form A1. In anotherembodiment, the transformation rate is accelerated in the temperaturerange of about 20° C. to about 200° C. In yet another embodiment, thetemperature ranges from about 30° C. to about 100° C.

According to another embodiment, the present invention provides a methodof converting polymorph Form B2 or Form C3 to Form A1 by exposing asolution or slurry of Form B2 or Form C3 in a suitable solvent ormixtures of solvents to a suitable temperature for a suitable period oftime and then cooling the slurry or solution and finally collecting thesolid Form A1. In another embodiment said suitable solvent or mixture ofsolvents is selected from those solvents indicated in Table 1 hereinbelow.

According to another embodiment, the present invention provides a methodof converting mixtures of either Form A1 and B2, or Form A1 and C3, orForm B2 and C3 to Form A1 by heating at a suitable temperature forsuitable period of time in a suitable solvent or mixture of solventswith suitable agitation. In one embodiment said suitable solvent(s)comprise ethyl acetate, 10% water/dioxane, 25% water/ethanol, ortetrahydrofuran, said suitable period of time is about 24 hours, andsaid suitable agitation comprises shaking.

According to another embodiment, the present invention provides apharmaceutical composition comprising Form A1 and a pharmaceuticallyacceptable carrier or adjuvant.

According to another embodiment, the present invention provides a methodof formulating a pharmaceutical composition comprising an amorphous formof Compound 1, comprising the steps of:

-   (i) converting Form A1 to an amorphous form; and-   (ii) combining said amorphous form with one or more suitable    pharmaceutical carrier or adjuvant.

According to another embodiment, the present invention provides a methodof treating an IMPDH-mediated disease in a patient comprising the stepof administering to said patient a therapeutically effective amount ofForm A1 or a pharmaceutical composition comprising Form A1.

According to another embodiment, the present invention provides aprocess for preparing a polymorph of Form A1, said process comprising atleast one of the following steps:

-   -   a) dissolving (S)-tetrahydrofuran-3-yl        3-(3-(3-methoxy-4-(oxazol-5-yl)phenyl)ureido)benzylcarbamate in        a suitable solvent with suitable agitation at a suitable        temperature to give a suitable solution;    -   b) adding a suitable volume of a suitable solvent over a        suitable period of time, at a suitable temperature with        agitation to generate a slurry;    -   c) cooling said slurry to a suitable temperature;    -   d) adding a suitable amount of the same solvent from step (b) to        the slurry;    -   e) cooling said slurry to about room temperature;    -   f) filtering or centrifuging said slurry to give polymorph Form        A;    -   g) rinsing said Form A a suitable number of times with a        suitable solvent; and    -   h) drying said Form A at a suitable temperature under a suitable        reduced pressure for a suitable period of time to constant        weight.

According to another embodiment, the present invention provides apolymorphic Form B2 of (S)-tetrahydrofuran-3-yl3-(3-(3-methoxy-4-(oxazol-5-yl)phenyl)ureido)benzylcarbamate.

According to another embodiment, the present invention provides apolymorphic Form B2 wherein the polymorph has a peak position at about20.9 degrees 2-theta in an x-ray powder diffraction pattern obtainedusing Cu K alpha radiation.

According to another embodiment, the present invention provides apolymorphic Form B2 wherein the polymorph has at least one additionalpeak position at about 5.3, 15.7, 18.4, or 20.0 degrees 2-theta in anx-ray powder diffraction pattern obtained using Cu K alpha radiation.

According to another embodiment, the present invention provides apolymorphic Form B2 wherein the polymorph exhibits a broad endothermicevent between about 80° C. and about 100° C. as measured by aDifferential Scanning Calorimeter.

According to another embodiment, the present invention provides apolymorphic Form B2 wherein the polymorph exhibits amelting/recrystallization event at between about 146° C. and about 150°C. as measured by a Differential Scanning Calorimeter.

According to another embodiment, the present invention provides apolymorphic Form B2 wherein the polymorph exhibits amelting/decomposition endothermic event at about 215° C. as measured bya Differential Scanning Calorimeter.

According to another embodiment, polymorphic Form B2 is a crystallineform containing various solvent levels. DSC analysis shows three events;a broad, weak endotherm between 80° C. and 100° C. which is consistentwith loss of solvent from the sample or a solid phase transition; amelting/re-crystallization event at about 146° C. to about 150° C.; anda final melting/decomposition endotherm with an onset of about 215° C.Microscopic analysis of Form B2 showed it to contain particles of size30-75 μm and showed bi-refringence.

According to another embodiment, the present invention provides aprocess for preparing Form B2, as exemplified herein below.

According to another embodiment, the present invention provides apharmaceutical composition comprising Form B2 and a pharmaceuticallyacceptable carrier or adjuvant.

According to another embodiment, the present invention provides a methodof formulating a pharmaceutical composition comprising an amorphous formof Compound 1, comprising the steps of:

-   (i) converting Form B2 to an amorphous form; and-   (ii) combining said amorphous form with one or more suitable,    pharmaceutical carrier or adjuvant.

According to another embodiment, the present invention provides a methodof treating an IMPDH-mediated disease in a patient comprising the stepof administering to said patient a therapeutically effective amount ofForm B2 or a pharmaceutical composition comprising Form B2.

According to another embodiment, the present invention provides apolymorphic Form B2 of (S)-tetrahydrofuran-3-yl3-(3-(3-methoxy-4-(oxazol-5-yl)phenyl)ureido)benzylcarbamate.

According to another embodiment, the present invention provides apolymorphic Form B2 wherein the polymorph has a peak position at about20.9 degrees 2-theta in an x-ray powder diffraction pattern obtainedusing Cu K alpha radiation.

According to another embodiment, the present invention provides apolymorphic Form B2 wherein the polymorph has at least one additionalpeak position at about 5.3, 15.7, 18.4, or 20.0 degrees 2-theta in anx-ray powder diffraction pattern obtained using Cu K alpha radiation.

According to another embodiment, the present invention provides apolymorphic Form B2 wherein the polymorph exhibits a broad endothermicevent between about 80° C. and about 100° C. as measured by aDifferential Scanning Calorimeter.

According to another embodiment, the present invention provides apolymorphic Form B2 wherein the polymorph exhibits amelting/recrystallization event at between about 146° C. and about 150°C. as measured by a Differential Scanning Calorimeter.

According to another embodiment, the present invention provides apolymorphic Form B2 wherein the polymorph exhibits amelting/decomposition endothermic event at about 215° C. as measured bya Differential Scanning Calorimeter.

According to another embodiment, the present invention provides apolymorphic Form C3 of (S)-tetrahydrofuran-3-yl3-(3-(3-methoxy-4-(oxazol-5-yl)phenyl)ureido)benzylcarbamate.

According to another embodiment, the present invention provides apolymorphic Form C3 wherein the polymorph has a peak position at about20.7 degrees 2-theta in an x-ray powder diffraction pattern obtainedusing Cu K alpha radiation.

According to another embodiment, the present invention provides apolymorphic Form C3 wherein the polymorph has at least one additionalpeak position at about 5.2, 15.5, 17.5, or 22.5 degrees 2-theta in anx-ray powder diffraction pattern obtained using Cu K alpha radiation.

According to another embodiment, the present invention provides apolymorphic Form C3 wherein the polymorph exhibits amelting/recrystallization event at between about 145° C. and about 160°C. as measured by a Differential Scanning Calorimeter.

According to another embodiment, Form C3 is an anhydrous crystallineform of Compound 1. Thermal analysis of the sample indicates that itundergoes a melt/re-crystallization transition at about 145° C.-160° C.Variable temperature XRPD confirms the transition from Form C3 to FormA1 between about 140° C. and about 160° C. Form C3 is moderatelyhygroscopic and exhibits 3% weight uptake at 90% relative humidity at25° C.

According to another embodiment, the present invention provides aprocess for preparing Form C3, as exemplified herein below.

According to another embodiment, the present invention provides apharmaceutical composition comprising Form C3 and a pharmaceuticallyacceptable carrier or adjuvant.

According to another embodiment, the present invention provides a methodof formulating a pharmaceutical composition comprising an amorphous formof Compound 1, comprising the steps of:

-   (i) converting Form C3 to an amorphous form; and-   (ii) combining said amorphous form with one or more suitable    pharmaceutical carrier or adjuvant.

According to another embodiment, the present invention provides a methodof treating an IMPDH-mediated disease in a patient comprising the stepof administering to said patient a therapeutically effective amount ofForm C3 or a pharmaceutical composition comprising Form C3.

According to another embodiment, the present invention provides apolymorphic Form D4 of (S)-tetrahydrofuran-3-yl3-(3-(3-methoxy-4-(oxazol-5-yl)phenyl)ureido)benzylcarbamate, HCl salt.

According to another embodiment, the present invention provides apolymorphic Form D4 wherein the polymorph has a peak position at about24.94 degrees 2-theta in an x-ray powder diffraction pattern obtainedusing Cu K alpha radiation.

According to another embodiment, the present invention provides apolymorphic Form D4 wherein the polymorph has at least one additionalpeak position at about 11.1, 15.7, 16.9, 18.8, or 27.4 degrees 2-thetain an x-ray powder diffraction pattern obtained using Cu K alpharadiation.

According to another embodiment, the present invention provides apolymorphic Form D4 wherein the polymorph exhibits a broad endothermicevent between about 100° C. and about 170° C. as measured by aDifferential Scanning Calorimeter.

According to another embodiment, Form D4 is a weakly crystalline HClsalt form of Compound 1. Microscopic analysis of Form D4 shows littlebirefringence typical of a weakly crystalline sample and upon heating ofthe sample a melt/decomposition is observed followed by are-crystallization of the material. This change is confirmed by variabletemperature XRPD analysis which shows a change in the pattern between140° C. and 160° C. to an XRPD pattern that is similar to Form A1 of thefree base. This is consistent with loss of the HCl from the salt andre-crystallization of the free base from the melt. Thermogravimetricanalysis of the salt shows a weight loss of 6.5% up to 170° C. which isapproximately (7.4% theoretical) the expected weight loss for loss ofHCl from the salt. This loss of HCl from the sample manifests itself asa broad endotherm in the DSC from about 100° C. to about 170° C.

According to another embodiment, the present invention provides aprocess for preparing Form D4 as exemplified herein below.

According to another embodiment, the present invention provides apharmaceutical composition comprising Form D4 HCl salt and apharmaceutically acceptable carrier or adjuvant.

According to another embodiment, the present invention provides a methodof formulating a pharmaceutical composition comprising an amorphous formof Compound 1, HCl salt, comprising the steps of:

-   (i) converting Form D4 to an amorphous form; and-   (ii) combining said amorphous form with one or more suitable    pharmaceutical carrier or adjuvant.

According to another embodiment, the present invention provides a methodof treating an IMPDH-mediated disease in a patient comprising the stepof administering to said patient a therapeutically effective amount ofForm D4 HCl salt or a pharmaceutical composition comprising Form D4 HClsalt.

According to another embodiment, the present invention provides apolymorphic Form E5 of (S)-tetrahydrofuran-3-yl3-(3-(3-methoxy-4-(oxazol-5-yl)phenyl)ureido)benzylcarbamate, HCl salt.

According to another embodiment, the present invention provides apolymorphic Form E5 wherein the polymorph has at least one peak positionat about 14.8 degrees 2-theta in an x-ray powder diffraction patternobtained using Cu K alpha radiation.

According to another embodiment, the present invention provides apolymorphic Form E5 wherein the polymorph has at least one additionalpeak position at about 19.4, 21.4, 22.5, or 25.4 degrees 2-theta in anx-ray powder diffraction pattern obtained using Cu K alpha radiation.

According to another embodiment, polymorphic Form E5 as an HCl salt, isa weakly crystalline salt form of Compound 1. Microscopic analysis ofForm E5 shows little bi-refringence typical of a weakly crystallinesample and upon heating of the sample a melt/decomposition is observedfollowed by a re-crystallization of the material. The variabletemperature XRPD analysis that shows a change in the pattern betweenabout 120° C. and about 150° C. confirms this change. This is consistentwith loss of the HCl from the salt and re-crystallization of the freebase from the melt. Form E5 HCl salt is highly hygroscopic with anuptake of 22% w/w at 90% relative humidity. DSC analysis of the sampleshows initial loss of water/solvent from the sample followed by meltingand loss of HCl. The latter two events are confirmed by hot stagemicroscopy and thermogravimetric/variable temperature-XRPD.

According to another embodiment, the present invention provides aprocess for preparing Form E5 HCl salt, as exemplified herein below.

According to another embodiment, the present invention provides apharmaceutical composition comprising Form E5 HCl salt and apharmaceutically acceptable carrier or adjuvant.

According to another embodiment, the present invention provides a methodof formulating a pharmaceutical composition comprising an amorphous formof Compound 1, comprising the steps of:

-   (i) converting Form E5 HCl salt to an amorphous form; and-   (ii) combining said amorphous form with one or more suitable    pharmaceutical carrier or adjuvant.

According to another embodiment, the present invention provides a methodof treating an IMPDH-mediated disease in a patient comprising the stepof administering to said patient a therapeutically effective amount ofForm E5 HCl salt or a pharmaceutical composition comprising Form E5 HClsalt.

According to another embodiment, the present invention provides asubstantially pure amorphous form of (S)-tetrahydrofuran-3-yl3-(3-(3-methoxy-4-(oxazol-5-yl)phenyl)ureido)benzylcarbamate whereinsaid amorphous Form comprises less than 5% by weight of Form A1.

According to another embodiment, the present invention provides anamorphous form of (S)-tetrahydrofuran-3-yl3-(3-(3-methoxy-4-(oxazol-5-yl)phenyl)ureido)benzylcarbamate.

According to another embodiment, the present invention provides aprocess to prepare an amorphous form of Compound 1 from Form B2 or FormC3 by evaporation of a solution of polymorphic Form B2 or Form C3 in asuitable solvent or a mixture of suitable solvents.

According to another embodiment, the present invention provides aprocess to prepare an amorphous Form HCl salt of Compound 1 from Form D4HCl salt or Form E5 HCl salt by evaporation of a solution of Form D4 HClsalt or Form E5 HCl salt in a suitable solvent or a mixture of suitablesolvents.

According to another embodiment, the present invention provides aprocess to prepare an amorphous form of Compound 1 from polymorphic FormA1 by evaporation of a solution of Form A1 in 2,2,2-trifluoroethanol orhexafluoroisopropanol or mixtures thereof.

According to another embodiment, the present invention provides aprocess to prepare an amorphous form of Compound 1 by cooling a moltensample of a crystalline form of (S)-tetrahydrofuran-3-yl3-(3-(3-methoxy-4-(oxazol-5-yl)phenyl)ureido)benzylcarbamate. Accordingto one embodiment, the amorphous form of Compound 1 is produced byconverting a crystalline form of Compound 1, e.g., Form A1, into anamorphous form of Compound 1.

According to another embodiment, the present invention provides apharmaceutical composition comprising the amorphous Form according toany of the embodiments herein, wherein said composition is produced bycombining said amorphous form with one or more suitable pharmaceuticalcarriers or adjuvants.

According to another embodiment, the present invention provides apharmaceutical composition comprising an amorphous form of Compound 1and a pharmaceutical acceptable adjuvant or carrier.

According to one embodiment, the present invention provides a method oftreating an IMPDH-mediated disease or condition in a mammal comprisingthe step of administering to said mammal a pharmaceutical compositioncontaining Form A1, Form B2, Form C3, Form D4, Form E5 or amorphousform.

According to another embodiment, the present invention provides a methodof treating an IMPDH-mediated disease or condition in a mammalcomprising the step of administering to said mammal a pharmaceuticalcomposition containing Form A1, Form B2, Form C3, Form D4, Form E5 oramorphous form.

According to another embodiment, the present invention provides a methodfor inhibiting viral replication in a mammal comprising the step ofadministering to said mammal a pharmaceutical composition containingForm A1, Form B2, Form C3, Form D4, Form E5 or amorphous form.

According to another embodiment, the present invention provides a methodfor treating a mammal suffering from a viral infection caused by a virusselected from hepatitis B virus, hepatitis C virus, orthomyxovirus,paramyxovirus, herpesvirus, retrovirus, flavivirus, pestivirus,hepatotrophic virus, bunyavirus, Hantaan virus, Caraparu virus, humanpapilloma virus, encephalitis virus, arena virus, reovirus, vesicularstomatitis virus, rhinovirus, entervirus, Lassa fever virus, togavirus,poxvirus, adenovirus, rubiola, or rubella.

Specific acid salts useful for producing salt forms of Compound 1, FormA1, Form B2, Form C3, and amorphous form and for producing salts otherthan the HCl salt for Form D4 and E5 may be selected from acids known inthe art. See, e.g., “Practical Process, Research & Development,”Anderson, Neal G., Academic Press, 2000, the contents of which isincorporated herein by reference.

The term “suitable” as used herein, describes solvent, temperature,filtrate, agitation, solution, medium, quantity, period of time, etc.Such suitable solvents, temperature, filtrate, agitation, solution,medium, quantity, period of time, etc. are readily known to one of skillin the art.

The term “pharmaceutically acceptable carrier or adjuvant” refers to anon-toxic carrier, adjuvant, or vehicle that does not destroy thepharmacological activity of the compound with which it is formulated.Pharmaceutically acceptable carriers, adjuvants or vehicles that may beused in the compositions of this invention include, but are not limitedto, ion exchangers, alumina, aluminum stearate, lecithin, serumproteins, such as human serum albumin, buffer substances such asphosphates, glycine, sorbic acid, potassium sorbate, partial glyceridemixtures of saturated vegetable fatty acids, water, salts orelectrolytes, such as protamine sulfate, disodium hydrogen phosphate,potassium hydrogen phosphate, sodium chloride, zinc salts, colloidalsilica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-basedsubstances, polyethylene glycol, sodium carboxymethylcellulose,polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers,polyethylene glycol and wool fat.

Pharmaceutically acceptable salts of the compounds of this inventioninclude those derived from pharmaceutically acceptable inorganic andorganic acids and bases. Examples of suitable acid salts includeacetate, adipate, alginate, aspartate, benzoate, benzenesulfonate,bisulfate, butyrate, citrate, camphorate, camphorsulfonate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,formate, fumarate, glucoheptanoate, glycerophosphate, glycolate,hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide,hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oxalate,palmoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, salicylate, succinate, sulfate, tartrate,thiocyanate, tosylate and undecanoate. Other acids, such as oxalic,while not in themselves pharmaceutically acceptable, may be employed inthe preparation of salts useful as intermediates in obtaining thecompounds of the invention and their pharmaceutically acceptable acidaddition salts.

Salts derived from appropriate bases include alkali metal (e.g., sodiumand potassium), alkaline earth metal (e.g., magnesium), ammonium andN⁺(C₁₋₄ alkyl)₄ salts. This invention also envisions the quaternizationof any basic nitrogen-containing groups of the compounds disclosedherein. Water or oil-soluble or dispersible products may be obtained bysuch quaternization.

The compositions of the present invention may be administered orally,parenterally, by inhalation spray, topically, rectally, nasally,buccally, vaginally or via an implanted reservoir. The term “parenteral”as used herein includes subcutaneous, intravenous, intramuscular,intra-articular, intra-synovial, intrasternal, intrathecal,intrahepatic, intralesional and intracranial injection or infusiontechniques. Preferably, the compositions are administered orally,intraperitoneally or intravenously. Sterile injectable forms of thecompositions of this invention may be aqueous or oleaginous suspension.These suspensions may be formulated according to techniques known in theart using suitable dispersing or wetting agents and suspending agents.The sterile injectable preparation may also be a sterile injectablesolution or suspension in a non-toxic parenterally-acceptable diluent orsolvent, for example as a solution in 1,3-butanediol. Among theacceptable vehicles and solvents that may be employed are water,Ringer's solution and isotonic sodium chloride solution. In addition,sterile, fixed oils are conventionally employed as a solvent orsuspending medium.

For this purpose, any bland fixed oil may be employed includingsynthetic mono- or di-glycerides. Fatty acids, such as oleic acid andits glyceride derivatives are useful in the preparation of injectables,as are natural pharmaceutically-acceptable oils, such as olive oil orcastor oil, especially in their polyoxyethylated versions. These oilsolutions or suspensions may also contain a long-chain alcohol diluentor dispersant, such as carboxymethyl cellulose or similar dispersingagents that are commonly used in the formulation of pharmaceuticallyacceptable dosage forms including emulsions and suspensions. Othercommonly used surfactants, such as Tweens, Spans and other emulsifyingagents or bioavailability enhancers which are commonly used in themanufacture of pharmaceutically acceptable solid, liquid, or otherdosage forms may also be used for the purposes of formulation.

The pharmaceutical compositions of this invention may be orallyadministered in any orally acceptable dosage form including, but notlimited to, capsules, tablets, aqueous suspensions or solutions. In thecase of tablets for oral use, carriers commonly used include lactose andcorn starch. Lubricating agents, such as magnesium stearate, are alsotypically added. For oral administration in a capsule form, usefuldiluents include lactose and dried cornstarch. When aqueous suspensionsare required for oral use, the active ingredient is combined withemulsifying and suspending agents. If desired, certain sweetening,flavoring or coloring agents may also be added.

Alternatively, the pharmaceutical compositions of this invention may beadministered in the form of suppositories for rectal administration.These can be prepared by mixing the agent with a suitable non-irritatingexcipient that is solid at room temperature but liquid at rectaltemperature and therefore will melt in the rectum to release the drug.Such materials include cocoa butter, beeswax and polyethylene glycols.

The pharmaceutical compositions of this invention may also beadministered topically, especially when the target of treatment includesareas or organs readily accessible by topical application, includingdiseases of the eye, the skin, or the lower intestinal tract. Suitabletopical formulations are readily prepared for each of these areas ororgans.

Topical application for the lower intestinal tract can be effected in arectal suppository formulation (see above) or in a suitable enemaformulation. Topically-transdermal patches may also be used.

For topical applications, the pharmaceutically acceptable compositionsmay be formulated in a suitable ointment containing the active componentsuspended or dissolved in one or more carriers. Carriers for topicaladministration of the compounds of this invention include, but are notlimited to, mineral oil, liquid petrolatum, white petrolatum, propyleneglycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax andwater. Alternatively, the pharmaceutically acceptable compositions canbe formulated in a suitable lotion or cream containing the activecomponents suspended or dissolved in one or more pharmaceuticallyacceptable carriers. Suitable carriers include, but are not limited to,mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax,cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

For ophthalmic use, the pharmaceutically acceptable compositions may beformulated as micronized suspensions in isotonic, pH adjusted sterilesaline, or, preferably, as solutions in isotonic, pH adjusted sterilesaline, either with or without a preservative such as benzylalkoniumchloride. Alternatively, for ophthalmic uses, the pharmaceuticallyacceptable compositions may be formulated in an ointment such aspetrolatum.

The pharmaceutically acceptable compositions of this invention may alsobe administered by nasal aerosol or inhalation. Such compositions areprepared according to techniques well-known in the art of pharmaceuticalformulation and may be prepared as solutions in saline, employing benzylalcohol or other suitable preservatives, absorption promoters to enhancebioavailability, fluorocarbons, and/or other conventional solubilizingor dispersing agents.

In one embodiment, the pharmaceutically acceptable compositions of thisinvention are formulated for oral administration.

The amount of the compounds of the present invention that may becombined with the carrier materials to produce a composition in a singledosage form will vary depending upon the host treated, the particularmode of administration. Preferably, the compositions should beformulated so that a dosage of between 0.01-100 mg/kg body weight/day ofthe compound can be administered to a patient receiving thesecompositions.

It should also be understood that a specific dosage and treatmentregimen for any particular patient will depend upon a variety offactors, including the activity of the specific compound employed, theage, body weight, general health, sex, diet, time of administration,rate of excretion, drug combination, and the judgment of the treatingphysician and the severity of the particular disease being treated. Theamount of a compound of the present invention in the composition willalso depend upon the particular compound in the composition.

Depending upon the particular condition, or disease, to be treated orprevented, additional therapeutic agents, which are normallyadministered to treat or prevent that condition, may also be present inthe compositions of this invention. As used herein, additionaltherapeutic agents that are normally administered to treat or prevent aparticular disease, or condition, are known as “appropriate for thedisease, or condition, being treated.”

When the compositions of this invention comprise a combination of anIMPDH inhibitor of this invention and one or more additional therapeuticor prophylactic agents, such as those disclosed herein, both the IMPDHinhibitor and the additional agent(s) should be present at dosage levelsof between about 10 to 100%, and more preferably between about 10 to 80%of the dosage normally administered in a monotherapy regimen. Theadditional agents may be administered separately, as part of a multipledose regimen, from the compounds of this invention. Alternatively, thoseagents may be part of a single dosage form, mixed together with thecompounds of this invention in a single composition.

When the compositions of this invention comprise a combination of anIMPDH inhibitor of this invention and one or more additional therapeuticor prophylactic agents, both the compound and the additional agentshould be present at dosage levels of between about 10 to 100% and inanother embodiment between about 10 to 80% of the dosage normallyadministered in a monotherapy regimen.

According to one embodiment, the pharmaceutical compositions of thisinvention comprise an additional immunosuppression agent. Examples ofadditional immunosuppression agents include, but are not limited to,cyclosporin A, FK506, rapamycin, leflunomide, deoxyspergualin,prednisone, azathioprine, mycophenolate mofetil, OKT3, ATAG, mizoribine,and interferon including alpha-interferon such as PEG-Intron® andPegasys®.

The term “interferon” as used herein means a member of a family ofhighly homologous species-specific proteins that inhibit viralreplication and cellular proliferation, and modulate immune response,such as interferon alpha, interferon beta, or interferon gamma. TheMerck Index, entry 5015, Twelfth Edition.

According to one embodiment of the present invention, the interferon isa-interferon. According to another embodiment, a therapeutic combinationof the present invention utilizes natural alpha interferon 2a. Or, thetherapeutic combination of the present invention utilizes natural alphainterferon 2b. In another embodiment, the therapeutic combination of thepresent invention utilizes recombinant alpha interferon 2a or 2b. In yetanother embodiment, the interferon is pegylated alpha interferon 2a or2b. Interferons suitable for the present invention include:

-   -   (a) Intron (interferon-alpha 2B, Schering Plough),    -   (b) Peg-Intron,    -   (c) Pegasys,    -   (d) Roferon,    -   (e) Berofor,    -   (f) Sumiferon,    -   (g) Wellferon,    -   (h) consensus alpha interferon available from Amgen, Inc.,        Newbury Park, Calif.,    -   (i) Alferon;    -   (j) Viraferon®; and    -   (k) Infergen®.

According to an alternate embodiment, the pharmaceutical compositions ofthis invention may additionally comprise an anti-cancer agent. Examplesof anti-cancer agents include, but are not limited to, cis-platin,actinomycin D, doxorubicin, vincristine, vinblastine, etoposide,amsacrine, mitoxantrone, tenipaside, taxol, colchicine, cyclosporin A,phenothiazines, interferon and thioxantheres.

In another embodiment, the compositions of this invention additionallycomprise another anti-viral agent, including an anti-HCV agent. Suchanti-viral agents include, but are not limited to, immunomodulatoryagents, such as α-, β-, and γ-interferons, pegylated derivatizedinterferon-α compounds, and thymosin; other anti-viral agents, such asribavirin (and the combination therapy of ribavirin and pegylatedinterferon [Rebetrol®]), d4T, ddI, AZT, amprenavir, fos-amprenavir,acyclovir, NS3-NS4A protease inhibitors such as those disclosed in PCTpublication No. WO 02/018369, amantadine, cytovene, ganciclovir,ritonivir, trisodium phosphonoformate, and telbivudine; other inhibitorsof hepatitis C proteases (NS2-NS3 inhibitors and NS3-NS4A inhibitors);inhibitors of other targets in the HCV life cycle, including but notlimited to, helicase and polymerase inhibitors; inhibitors of internalribosome entry; and broad-spectrum viral inhibitors, such as IMPDHinhibitors (e.g., IMPDH inhibitors disclosed in U.S. Pat. Nos. 5,807,876and 6,498,178, mycophenolic acid and derivatives thereof).

In another embodiment, the compositions of this invention additionallycomprise another anti-viral agent selected from alpha-interferon,pegylated alpha-interferon, or ribavirin.

In one embodiment, the compositions of this invention additionallycomprise another agent, including a cytochrome P-450 inhibitor. Suchcytochrome P-450 inhibitors include, but are not limited to, ritonavir.CYP inhibitors may be useful in increasing liver concentrations and/orincreasing blood levels of compounds that are inhibited by CYP.

If an embodiment of this invention involves a CYP inhibitor, any CYPinhibitor that improves the pharmacokinetics of the IMPDH inhibitor maybe used in a method of this invention. These CYP inhibitors include, butare not limited to, ritonavir (WO 94/14436), ketoconazole,troleandomycin, 4-methyl pyrazole, cyclosporin, clomethiazole,cimetidine, itraconazole, fluconazole, miconazole, fluvoxamine,fluoxetine, nefazodone, sertraline, indinavir, nelfinavir, amprenavir,fosamprenavir, saquinavir, lopinavir, delavirdine, erythromycin, VX-944,and VX-497. Preferred CYP inhibitors include ritonavir, ketoconazole,troleandomycin, 4-methyl pyrazole, cyclosporin, and clomethiazole. Forpreferred dosage forms of ritonavir, see U.S. Pat. No. 6,037,157, andthe documents cited therein: U.S. Pat. No 5,484,801, U.S. applicationSer. No. 08/402,690, and International Applications WO 95/07696 and WO95/09614).

Methods for measuring the ability of a compound to inhibit cytochromeP450 monooxygenase activity are known (see U.S. Pat No. 6,037,157 andYun, et al. Drug Metabolism & Disposition, vol. 21, pp. 403-407 (1993).

According to yet another alternate embodiment, the pharmaceuticalcompositions of this invention may additionally comprise ananti-vascular hyperproliferative agent. Examples of anti-vascularhyperproliferative agents include, but are not limited to, HMG Co-Areductase inhibitors such as lovastatin, thromboxane A2 synthetaseinhibitors, eicosapentanoic acid, ciprostene, trapidil, ACE inhibitors,low molecular weight heparin, mycophenolic acid, rapamycin and5-(3′-pyridinylmethyl)benzofuran-2-carboxylate.

Upon improvement of a patient's condition, a maintenance dose of acompound, composition or combination of this invention may beadministered, if necessary. Subsequently, the dosage or frequency ofadministration, or both, may be reduced, as a function of the symptoms,to a level at which the improved condition is retained when the symptomshave been alleviated to the desired level, treatment should cease.Patients may, however, require intermittent treatment on a long-termbasis upon any recurrence of disease symptoms.

According to one embodiment, the term “IMPDH-mediated disease” as usedherein includes immune system related diseases such as transplantrejection (e.g., kidney, liver, heart, lung, pancreas (islet cells),bone marrow, cornea, small bowel and skin allografts and heart valvexenografts), graft versus host disease, and autoimmune diseases, such asrheumatoid arthritis, multiple sclerosis, juvenile diabetes, asthma,inflammatory bowel disease (Crohn's disease, ulcerative colitus), lupus,diabetes, mellitus myasthenia gravis, psoriasis, dermatitis, eczema,seborrhea, pulmonary inflammation, eye uveitis, Grave's disease,Hashimoto's thyroiditis, Behcet's or Sjorgen's syndrome (dryeyes/mouth), pernicious or immunohaemolytic anaemia, idiopathic adrenalinsufficiency, polyglandular autoimmune syndrome, glomerulonephritis,scleroderma, lichen planus, viteligo (depigmentation of the skin),autoimmune thyroiditis, and alveolitis.

According to another embodiment, the term “IMPDH-mediated disease” asused herein includes viral diseases such as DNA and RNA viral diseasescaused by infection for example, by orthomyxoviruses (influenza virusestypes A and B), paramyxoviruses (respiratory syncytial virus (RSV),subacute sclerosing panencephalitis (SSPE) virus) measles andparainfluenza type 3), herpesviruses (HSV-1, HSV-2, HHV-6, HHV-7, HHV-8,Epstein Barr Virus (EBV), cytomegalovirus (HCMV) and varicella zostervirus (VZV)), retroviruses (HIV-1, HIV-2, HTLV-1, HTLV-2), flavi- andpestiviruses (yellow fever virus (YFV), hepatitis C virus (HCV), denguefever virus, bovine viral diarrhea virus (BVDV), hepatotrophic viruses(hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis D virus(HDV), hepatitis E virus (HEV), hepatitis G virus (HGV), Crimean-Congohemorrhagic fever virus (CCHF), bunyaviruses (Punta Toro virus, RiftValley fever virus (RVFV), and sandfly fever Sicilian virus), Hantaanvirus, Caraparu virus), human papilloma viruses, encephalitis viruses(La Crosse virus), arena viruses (Junin and Tacaribe virus), reovirus,vesicular stomatitis virus, rhinoviruses, enteroviruses (polio virus,coxsackie viruses, encephalomyocarditis virus (EMC)), Lassa fever virus,and togaviruses (Sindbis and Semlike forest viruses) and poxviruses(vaccinia virus), adenoviruses, rubiola, and rubella.

According to another embodiment, the term “IMPDH-mediated disease” asused herein includes vascular cellular hyperproliferative diseases suchas restenosis, stenosis, artherosclerosis and other hyperproliferativevascular disease.

According to another embodiment, the term “IMPDH-mediated disease” asused herein includes tumors and malignancies, such as lymphoma, leukemiaand other forms of cancer such as breast cancer, prostate cancer, coloncancer, pancreatic cancer, etc.

According to another embodiment, the term “IMPDH-mediated disease” asused herein includes inflammatory diseases such as osteoarthritis, acutepancreatitis, chronic pancreatitis, asthma and adult respiratorydistress syndrome.

Suitable methods for the conversion of a crystalline form, such as apolymorphic form of the present invention, into an amorphous formsuitable for formulation are well known in the art. See, e.g.,“Remington: The Science & Practice of Pharmacy”; Alfonso R. Gennaro,Editor, Mack Publishing, 1995, 19th Edition, Volume 2, the entiredisclosure whereof is incorporated herein by reference.

In order that the invention described herein may be more fullyunderstood, the following experimental methods, assays, and examples areset forth. The following experimental methods, assays, and examples areoffered by way of illustration, not limitation.

Experimental Methods

X-RAY Powder Diffraction (XRPD)

X-ray powder diffraction patterns for the samples were acquired on aBruker AXS/Siemens D5000 diffractometer using CuKα radiation (40 kV, 40mA), ?-? goniometer, automatic divergence and receiving slits, agraphite secondary monochromator and a scintillation counter. The datawere collected over an angular range of 2° to 42° 2? in continuous scanmode using a step size of 0.02° 2? and a step time of 1 second. Samplesrun under ambient conditions were prepared as flat plate specimens usingpowder as received without grinding. Approximately 25-50 mg of thesample was gently packed into 12 mm diameter, 0.5 mm deep cavities cutinto polished, zero-background (510) silicon wafers (The Gem Dugout,1652 Princeton Drive, Pennsylvania State College, PA 16803, USA). Allspecimens were run both stationary and rotated in their own plane duringanalysis. A further specimen was run using silicon powder as an internalstandard to correct for any peak displacement. Samples run undernon-ambient conditions were packed into a stainless steel cavity sampleholder equipped with a Pt 100 thermocouple for temperature monitoring.Low temperature data were recorded using an Anton Paar TTK450 variabletemperature camera attached to the Bruker AXS/Siemens D5000diffractometer. Instrumental conditions for the low temperature scanwere similar to those described for the flat plate samples above. AllXRPD analyses were performed using the Diffrac Plus XRD Commandersoftware v2.3.1. Diffraction data are reported using Cu Kα₁ (λ=1.5406Å), after the Kα₂ component had been stripped using EVA, the powderpatterns were indexed by the ITO method using WIN-INDEX and the rawlattice constants refined using WIN-METRIC.

Alternatively, X-ray powder diffraction patterns for the samples wereacquired on a Bruker AXS C2 GADDS diffractometer using Cu Kα radiation(40 kV, 40 mA), automated XYZ stage, laser video microscope forauto-sample positioning and a HiStar 2-dimensional area detector. X-rayoptics consists of a single Göbel multilayer mirror coupled with apinhole collimator of 0.3 mm. Beam divergence, i.e. the effective sizeof the X-ray beam on the sample, was approximately 4 mm. A ?-?continuous scan mode was employed with a sample to detector distance of20 cm which gives an effective 2? range of 3.2-29.8°. A typical exposuretime of a sample would be 120 s. Samples run under ambient conditionswere prepared as flat plate specimens using powder as received withoutgrinding. Approximately 1-2 mg of the sample was lightly pressed on aglass slide to obtain a flat surface. Samples run under non-ambientconditions were mounted on a silicon wafer with heat conductingcompound. The sample was then heated to the appropriate temperature atapproximately 20° C./minute and subsequently held isothermally forapproximately 1 minute before data collection was initiated

Differential Scanning Calorimetry (DSC)

Differential scanning calorimetry data was collected on a TA instrumentQ1000 equipped with a 50 position autosampler. The energy andtemperature calibration standard was indium. Samples were heated at arate of 10° C./min between 10 and 230° C. A nitrogen purge at 30 ml/minwas maintained over the sample. Between 1 and 3 mg of sample was used,unless otherwise stated, and all samples crimped in a hermeticallysealed aluminium pan.

Hot Stage Microscopy

Hot stage microscopy was studied using a Leica LM/DM polarisedmicroscope combined with a Mettler-Toledo MTFP82HT hot-stage in thetemperature range 25-230° C. with typical heating rates in the range10-20° C./min. A small amount of sample was dispersed onto a glass slidewith individual particles separated as well as possible. Samples wereviewed under normal or cross-polarised light (coupled to a λfalse-colour filter) with a ×20 objective lens.

Thermogravimetric Analysis (TGA)

TGA data was collected a TA Instrument Q500 TGA, calibrated withNickel/Alumel and running at scan rates of 10° C./minute. A nitrogenpurge at 60 ml/min was maintained over the sample. Typically 10-20 mg ofsample was loaded onto a pre-tared platinum crucible.

Infra-Red Spectroscopy by FT-IR (IR)

Samples were studied on a Perkin-Elmer Spectrum One fitted with aUniversal ATR sampling accessory. The data was collected and analysedusing Spectrum V5.0.1 software.

Fast Evaporation (FE)

Solutions were prepared in various solvents and sonicated betweenaliquot additions to assist in dissolution. Once a mixture reachedcomplete dissolution, as judged by visual observation, the solution wasfiltered through a 0.2 μm nylon filter. The filtered solution wasallowed to evaporate at ambient temperature in an open vial. The solidsthat formed were isolated and analyzed.

Slow Evaporation (SE)

Solutions were prepared in various solvents and sonicated betweenaliquot additions to assist in dissolution. Once a mixture reachedcomplete dissolution, as judged by visual observation, the solution wasfiltered through a 0.2 μm nylon filter. The filtered solution wasallowed to evaporate at ambient temperature in a vial covered withaluminum foil perforated with pinholes. The solids that formed wereisolated and analyzed.

Slow Cool (SC)

Saturated solutions were prepared in various solvents at elevatedtemperatures (approximately 60° C.) and filtered through a 0.2 μm nylonfilter into an open vial while still warm. The vial was covered andallowed to cool slowly to room temperature. The presence or absence ofsolids was noted. If there were no solids present, or if the amount ofsolids was judged too small for XRPD analysis, the vial was placed in arefrigerator overnight. Again, the presence or absence of solids wasnoted and if there were none, the vial was placed in a freezerovernight. Solids that formed were isolated by filtration and allowed todry prior to analysis.

Rotary Evaporation

Solutions were prepared in various solvents. The solution was thenfiltered into a round bottom flask and the solvent was removed by rotaryevaporation. Solids were recovered and analyzed.

Cold Precipitation

Solutions were prepared in various solvents at elevated temperature(approximately 60° C.) and filtered through a 0.2-μm nylon filter intoan antisolvent at sub-ambient temperature (an ice-water bath atapproximately 0° C. when water was used, and a dry ice/acetone bath atapproximately −78° C. for all other solvents). The resulting solids wereisolated by filtration and dried prior to analysis.

Slurry Experiments (SE)

Solutions were prepared by adding enough solids to a given solvent sothat undissolved solids were present. The mixture was then agitated in asealed vial at a given temperature. After a given amount of time, thesolids were isolated by suction filtration and analyzed.

In order that this invention be more fully understood, the followingexamples are offered by way of illustration, not limitation.

Assays

IMPDH Activity Inhibition Assay

IMP dehydrogenase activity was assayed following an adaptation of themethod first reported by Magasanik. [B. Magasanik et al., J. Biol.Chem., 226, p. 339 (1957), the disclosure of which is hereinincorporated by reference]. Enzyme activity was measuredspectrophotometrically, by monitoring the increase in absorbance at 340nm due to the formation of NADH (λ340 is 6220 M⁻¹ cm⁻¹). The reactionmixture contained 0.1 M potassium phosphate 8.0, 0.5 mM EDTA, 2 mM DTT,200 μM IMP and enzyme (IMPDH human type II) at a concentration of 15 to50 nM. This solution is incubated at 37° C. for 10 minutes. The reactionis started by adding NAD to a final concentration of 200 μM and theinitial rate is measured by following the linear increase in absorbanceat 340 nm for 10 minutes. For reading in a standard spectrophotometer(path length 1 cm) the final volume in the cuvette is 1.0 ml. The assayhas also been adapted to a 96 well microtiter plate format; in this casethe concentrations of all the reagents remain the same and the finalvolume is decreased to 200 μl. For the analysis of inhibitors, thecompound in question is dissolved in DMSO to a final concentration of 20mM and added to the initial assay mixture for preincubation with theenzyme at a final volume of 2-5% (v/v). The reaction is started by theaddition of NAD, and the initial rates measured as above. K_(i)determinations are made by measuring the initial velocities in thepresence of varying amounts of inhibitor and fitting the data using thetight-binding equations of Henderson (Henderson, P. J. F. (1972)Biochem. J. 127, 321).

Cellular Assays

A. Isolation of peripheral blood mononuclear cells (PBMCs): Human venousblood was drawn from normal healthy volunteers using heparin as ananti-coagulant. PBMCs were isolated from blood by centrifugation overFicoll-paque gradient or CPT tubes (Becton-Dickinson) using standardconditions. PBMCs were harvested, washed and re-suspended in completeRPMI, counted and diluted to 1×10⁶ cells/mL.

B. PBMC and splenocyte proliferation assays: 5×10⁴ cells (for human PBMCT cells) or 1×10⁵ cells (for human PBMC B cells) were added per well ofa 96-well plate. For T-cell assays, phyto-hemagglutinin (PHA) was addedto a final concentration of 10-20 μg/mL per well for cell. For B-cellassays, Staphylococcal protein A (SPAS) was added to a finalconcentration of 2 μg/mL per well. Serial 4-fold dilutions of inhibitorstocks were made in complete RPMI media and added to cells such that thefinal concentration of compounds ranged from 20 μM to 20 nM, while DMSOwas maintained at a final concentration of 0.1%. The cells were thenincubated for 3 days. All samples were tested in triplicate. Tritiatedthymidine (0.4 μCi/well) was added for the last 24 hours of the assay.The cells were harvested onto Betaplate filters and counted in ascintillation counter. Concentrations of compounds required to inhibitproliferation of cells by 50% (IC50 values) were calculated using theSoftMax Pro™ (Molecular Devices) computer software package.

Anti-Viral Assays

The anti-viral efficacy of compounds may be evaluated in various invitro and in vivo assays. For example, compounds may be tested in invitro viral replication assays. In vitro assays may employ whole cellsor isolated cellular components. In vivo assays include animal modelsfor viral diseases. Examples of such animal models include, but are notlimited to, rodent models for HBV or HCV infection, the Woodchuck modelfor HBV infection, and chimpanzee model for HCV infection.

Abbreviations and terms which are used in the examples that follow andthroughout the specification include:

EtOAc: ethyl acetate i-BuOAc: isobutyl acetate i-PrOAc: isopropylacetate MEK: methyl ethyl ketone MIBK: methyl isobutyl ketone TBME:tert-butyl methyl ether MeOH: methanol EtOH: ethyl alcohol TFE:2,2,2-trifluoroethanol IPA: isopropyl alcohol HFIPA:hexafluoroisopropanol ACN: acetonitrile THF: tetrahydrofuran NMP:N-methylpyrrolidinone DMF: N,N-dimethylformamide DMSO: dimethylsulfoxideHCl: hydrochloric acid N₂: nitrogen gas L: liter ml: milliliter T_(max):maximum temperature g: gram Kg: kilogram mg: milligram M: molar VT:variable temperature PBMC: peripheral blood mononuclear cells PHA:phyto-hemagglutinin SPAS: Staphylococcal protein A DTT: dithiothreitolEDTA: ethylenediaminetetraacetic acid NAD: nicotinamide adeninedinucleotide CPT: cell preparation tube RPMI: Roswell Park MemorialInstitute HBV: hepatitis B virus HCV: hepatitis C virus

EXAMPLES Example 1

Compound 1 used to prepare the polymorphs of this invention may besynthesized using the methods described in the Compound 1 patents. Inaddition, Compound 1 of this invention may be prepared by standardmanipulations of methods known to those skilled in the art

Example 2 Preparation of Form A1

1.8 g of Compound 1 in 3.6 ml of N-methylpyrrolidinone in a 50 ml roundbottom flask was heated to 60° C. Four volumes (7.2 ml) of methanol wasadded and crystallization soon became evident at 60° C. The suspensionwas cooled to 50° C., additional methanol (14.4 ml) was added and themixture finally cooled to room temperature and filtered. The filtratewas washed with methanol twice (3.6 ml each), then dried in vacuo togive 1.65 g of Form A1 (92% yield).

Example 3 Maturation Study of Form A1

Approximately 10 mg of Form A1 was slurried in 500 μl of solvent (seeTable 1 below) and shaken at 25-30° C. for 24 hrs. The excess solid wasremoved by filtration and analysed by XRPD. The results of the XRPDanalysis after 24 hrs are shown in Table 1 below. From all the 31solvents screened in Table 1, the XRPD patterns observed were consistentwith the pattern of Form A1.

TABLE 1 Expt No Solvent XRPD Pattern 1 Acetone A1 2 1-butanol A1 32-butanol A1 4 butyl acetate A1 5 TBME A1 6 EtOH A1 7 EtOAc A1 8 Ethylformate A1 9 heptane A1 10 i-BuOAc A1 11 i-PrOAc A1 123-methyl-1-butanol A1 13 MEK A1 14 MIBK A1 15 2-methyl-1-propanol A1 161-pentanol A1 17 1-propanol A1 18 IPA A1 19 propyl acetate A1 20 THF A121 ACN A1 22 Toluene A1 23 MeOH A1 24 10% H2O/MeOH A1 25 10% H2O/EtOH A126 10% H2O/IPA A1 27 10% H2O/ACN A1 28 10% H2O/THF A1 29 10% H2O/acetoneA1 30 10% H2O/dioxane A1 31 10% H2O/butanol A1

Example 4 Crystallization Experiments with Compound 1

Table 2 below provides a summary of crystallization experiments carriedout on Compound 1 using various solvents and conditions describedherein. The resulting Form A1 or amorphous form obtained were dried invacuo then analyzed by XRPD.

TABLE 2 Solvent Conditions^(a) Habit/Description XRPD Result^(b) acetoneslurry white solid A1 ACN slurry white solid A1 CH₂Cl₂ SC whitebirefringent fibers amorphous slurry off white solid A1 diethyl etherslurry white solid A1 DMF FE white residue, no A1 extinguishment SEwhite chunks, no A1 extinguishment 1,4-dioxane SC No solids — slurrywhite solid A1 EtOH SC white fibers A1 slurry white solid A1 EtOAcslurry white solid A1 HFIPA FE white residue, no amorphousextinguishment SE white residue, no amorphous extinguishment rotovap offwhite solid amorphous hexanes slurry white solid A1 IPA SC whiteirregular bits, no A1 birefringence slurry white solid A1 MeOH SCrosettes of white fibers A1 slurry white solid A1 THF FE whitedendridics, some A1 (LC) extinguishment SE — — off white solid A1 — —toluene slurry white solid A1 2,2,2-TFE FE — — irregulars, no A1extinguishment SE white round granules, no A1 extinguishment — — — — SCwhite chunks, no A1 (LC) birefringence trifluorotoluene slurry whitesolid A1 ^(a)FE = fast evaporation; SE = slow evaporation; SC = slowcool, rotovap = rotary evaporation ^(b)LC = low crystallinity;

Example 5 Preparation of Form B2 by Cold Precipitation

Form B2 was obtained from a cold precipitation experiment involving DMFas the solvent and toluene or ethyl acetate as the anti-solvent. Thereina solution of Compound 1 in DMF at approximately 60° C. was filteredthrough a 0.2-μm nylon filter into toluene at approximately −78° C. Theresulting solids were isolated by filtration and dried to give Form B2.XRPD analysis was consistent with Form B2.

Example 6 Preparation of Form B2 by Cold Precipitation

500 mg of Form A1 was dissolved in DMF (3.5 ml) at ambient temperatureto obtain a clear solution. The clear solution was filtered to removeany undissolved Form A1. The clear solution was added to ethyl acetate(150 ml) at −78° C. The resulting mixture was placed at −20° C. for 5days.

Precipitated solid was collected by filtration under vacuum and dried at40° C. in vacuo to give Form B2 as a white solid. XRPD analysis wasconsistent with Form B2.

Example 7 Preparation of Form C3 by Heating

A sample of Form C3 was generated by heating a sample of polymorph FormB2 at 100° C. for one hour. XRPD analysis was consistent with Form C3.

Example 8 Preparation of Form D4, HCl salt

30 mg of Compound 1 was dissolved in 2 ml of a 50:50 mix of 5M HCl inisopropyl alcohol and 1M HCl in acetic acid at room temperature. A smallportion of residual solid was removed by filtration. t-Butyl methylether (3 ml) was added to the clear solution and the sample placed at−20° C. overnight. The white solid present was collected by filtrationand then vacuum-dried at room temperature to give Form D4 as an HClsalt. XRPD analysis was consistent with Form D4, HCl salt.

Example 9 Preparation of Form D4, HCl salt

30 mg of Compound 1 was dissolved in 2 ml of a 50:50 mix of 5M HCl inisopropyl alcohol and 1M HCl in acetic acid at room temperature. A smallportion of residual solid was removed by filtration. t-Butyl methylether (3 ml) was added to the clear solution and the sample placed at−20° C. overnight. The white solid present was collected by filtrationand then vacuum-dried at room temperature to give Form D4 as an HClsalt. XRPD analysis was consistent with Form D4, HCl salt.

Example 10 Preparation of Form E5, HCl Salt

200 mg of Compound 1 was dissolved in 10 ml of 37.5% wt HCl solution.Water (10 ml) was added and a white precipitate was immediately evident.The slurry was placed at 0-5° C. and stirred for 1 hour. Precipitatedsolid was collected by filtration, air dried under vacuum for 2 hrs anddried in vacuo at 40° C. for 3 days to give Form E5 as an HCl salt. XRPDanalysis was consistent with Form E5, HCl salt.

Example 11 Preparation of Amorphous Material from Form A1

130 mg of Compound 1 (Form A1) was dissolved in 2,2,2-trifluoroethanol(6 ml) at room temperature to give a clear solution. Toluene was added(1 volume) and the solution was concentrated to dryness on a rotaryevaporator to give an amorphous solid. The sample was dried in vacuo for24 hrs at 40° C. After oven drying the sample was ground with a mortarand pestle to increase the amorphicity. XRPD analysis was consistentwith amorphous form.

1. A polymorphic Form A1 of (S)-tetrahydrofuran-3-yl3-(3-(3-methoxy-4-(oxazol-5-yl)phenyl)ureido)benzylcarbamate whereinsaid polymorph is characterized by peak positions at about 21.8, 11.8,16.0, 18.5, 20.1 and 23.6 degrees 2-theta in an x-ray powder diffractionpattern obtained using Cu K alpha radiation.
 2. The polymorphic Form A1according to claim 1, wherein the polymorph exhibits amelting/decomposition endothermic event at about 215° C. as measured bya Differential Scanning Calorimeter.
 3. A polymorphic Form A1of(S)-tetrahydrofuran-3-yl3-(3-(3-methoxy-4-(oxazol-5-yl)phenyl)ureido)benzylcarbamate whereinsaid polymorph exhibits a melting/decomposition endothermic event atabout 215° C. as measured by a Differential Scanning Calorimeter.
 4. Apolymorphic Form A1, wherein said polymorphic Form A1 has a peakposition at about 21.8 degrees 2-theta in an x-ray powder diffractionpattern obtained using Cu K alpha radiation.
 5. A polymorphic Form A1,wherein said polymorphic Form A1 has peak position at about 11.8 degrees2-theta in an x-ray powder diffraction pattern obtained using Cu K alpharadiation.
 6. A polymorphic Form A1, wherein said polymorphic Form A1has peak position at about 16.0 degrees 2-theta in an x-ray powderdiffraction pattern obtained using Cu K alpha radiation.
 7. Apolymorphic Form A1, wherein said polymorphic Form A1 has peak positionat about 18.5 degrees 2-theta in an x-ray powder diffraction patternobtained using Cu K alpha radiation.
 8. A polymorphic Form A1, whereinsaid polymorphic Form A1 has peak position at about 20.1 degrees 2-thetain an x-ray powder diffraction pattern obtained using Cu K alpharadiation.
 9. A polymorphic Form A1, wherein said polymorphic Form A1has peak position at about 23.6 degrees 2-theta in an x-ray powderdiffraction pattern obtained using Cu K alpha radiation.
 10. Apolymorphic Form A1, wherein said polymorphic Form A1 is characterizedby an x-ray powder diffraction pattern as shown in FIG. 1.